UNCOVERING A MISSING TRANSCRIPTOMIC CORNERSTONE OF ILEAL FIBROSTENOSIS IN CROHN’S DISEASE: HUB GENE NETWORKS AND CANDIDATE SERUM BIOMARKERS FROM AN INTEGRATIVE RNA-SEQ STUDY

Abstract BACKGROUND Fibrostenosis is a frequent and morbid complication of Crohn’s disease (CD), yet transcriptomic signatures of ileal stricturing remain poorly defined. Microarray studies provided limited gene coverage, while single-cell RNA-seq—although insightful—remains constrained by cost, technical complexity, and small sample sizes. Bulk RNA-seq of surgical biopsies captures the full multicellular microenvironment of the diseased ileum with greater statistical power and clinical relevance. To our knowledge, this is the first study integrating multiple independent bulk RNA-seq cohorts of adult ileal tissue to identify reproducible hub genes in fibrostenosing CD. METHODS Two independent RNA-seq datasets comprising 34 paired ileal samples from adults with stricturing CD were reprocessed with a harmonized DESeq2 pipeline (FDR < 0.05, |log2FC| ≥ 1.5, Ensembl/biomaRt annotation). Differentially expressed genes (DEGs) concordant across cohorts were intersected. Shared DEGs were uploaded to STRING (human interactome, medium confidence ≥0.4) to construct a protein–protein interaction (PPI) network. Hub genes were ranked with CytoHubba (MCC as primary metric; EPC/MNC as supportive). Pathway enrichment was assessed in Cytoscape. RESULTS Both cohorts yielded more than 1000 DEGs (padj < 0.05, |log2FC| ≥ 1.5). A total of 121 genes were consistently dysregulated across datasets, of which 82 were upregulated and 39 downregulated in strictured ileum. STRING network analysis of these common DEGs produced a highly connected PPI (119 nodes, 642 edges; enrichment p < 1e-16). Upregulated hubs included COL1A1, COL1A2, COL3A1, and FN1, highlighting extracellular matrix remodeling, while downregulated signals involved epithelial and metabolic regulators. Pathway enrichment identified ECM–receptor interaction, focal adhesion, PI3K–Akt signaling, and TGF-β signaling as the most significant modules. CONCLUSIONS This study represents the first integrative bulk RNA-seq analysis of whole surgical ileal biopsies in fibrostenosing Crohn’s disease, defining a conserved hub-gene network reproducible across independent cohorts. Beyond characterizing the fibrotic transcriptome, the findings highlight therapeutic relevance, as collagens and FN1 overlap with antifibrotic pipelines currently under development, including anti-integrin strategies, TGF-β modulators, and MMP inhibitors. Several hub genes, such as SPARC, IGFBP7, and VWF, encode secreted proteins and may hold promise as non-invasive serum biomarkers. Together, these hub genes and secreted proteins provide a rationale for the development of targeted therapies in fibrostenosing Crohn’s disease. Further studies leveraging multi-omics integration will be essential to expand these insights and ultimately advance precision medicine strategies for this highly morbid complication.